Use of rapamycin to inhibit response and induce tolerance to gene therapy vector and encoded transgene products

ABSTRACT

Disclosed are methods for transient co-administration of rapamycin together with a gene therapy vector encoding a transgene. The present invention is directed to inhibiting the immune response of a host to the administered gene therapy vector and encoded trans gene product, thus allowing persistent trans gene expression and repeated administration of the gene therapy product to the host. The present invention is also of relevance in genetic disease patients that mount immune responses to protein replacement therapies in which case the present invention provides for transient co-administration of rapamycin together with protein replacement therapy. In a further aspect of the invention, co-administration of rapamycin could inhibit a secondary immune response in a host that has been pre-immunized with the gene therapy vector or pre-immunized with the protein product encoded by the transgene.

The present application is a continuation of co-pending U.S. Ser. No.09/911,782 filed on Jul. 24, 2001 which is a continuation in part ofU.S. Ser. No. 09/876,574 filed on Jun. 7, 2001 which claims priority toU.S. Provisional Application No. 60/221,738, filed Jul. 31, 2000. Thecontents of the above-referenced applications are hereby incorporated byreference into the present disclosure.

BACKGROUND

Immunosuppressant drugs have been used for purposes of preventingadverse immune responses, either a rejection of a transplanted organ, oran attack on the patients own body by its own immune system caused by anautoimmune disease, without unduly suppressing the ability of thepatient's immune system to combat infection. Such immunosuppressantshave included rapamycin [U.S. Pat. No. 5,694,950]; FK 506 [U.S. Pat. No.5,365,948]; and cyclosporine.

With the advent of gene therapy, a need exists for methods of repeatadministration of gene therapy vectors, such as viral vectors, exists.Methods are needed which are able to effectively overcome the body'snormal immune response to gene therapy vectors such as viral vectors. Inorder to overcome the immunologic problems associated with repeatadministration of adenoviral vectors, the use of broadimmunosuppressants (Engelhardt et al., Proc. Natl. Acad. Sci. USA91:6196-6200 (1994)) and cytoablative agents (Dai et al., Proc. Natl.Acad. Sci. USA 92:1401-1405 (1995)) to overcome the immune response ofthe host to first generation Ad vectors have been tested. Transientco-administration of an immunoglobulin, CTLA4-Ig, along with anintravenous injection of Ad vector expressing a non-immunogenictransgene product (human oc-I anti-trypsin) has been shown to lead topersistent transgene expression from mouse liver (Kay et al., Nat.Genetics 11:191-197 (1995)). CTLA4-Ig blocks the B7-CD28 pathway of Tcell co-stimulation, which is required for optional activation of Tcells. (Jenkins et al., Immunity 1:443-446 (1994); Lenschow et al., Ann.Rev. Immunol. 14:233-258 (1996)). Although adenoviral-specific antibodylevels were reduced in CTLA4-Ig treated mice, the inhibition was notsufficient to allow secondary gene transfer via repeat administration ofthe vector under the conditions tested (Kay et al., Nat. Genetics11:191-197 (1995)). Thus, many immunosuppressant molecules are noteffective for gene therapy purposes in which persistent expression of aforeign transgene is desired. Accordingly, a need exists for methods ofemploying immunosuppressant drugs which are effective when used withgene therapy vectors.

SUMMARY OF INVENTION

The present invention provides for transient co-administration ofrapamycin together with a gene therapy vector encoding a transgene. Thepresent invention is directed to inhibiting the immune response of ahost to the administered gene therapy vector and to the encodedtransgene product, thus allowing persistent transgene expression andrepeated administration of the gene therapy product to the host. Thepresent invention is also of relevance in genetic disease patients thatmount immune responses to protein replacement therapies, in which casethe present invention provides for transient co-administration ofrapamycin together with protein replacement therapy. In a further aspectof the invention, co-administration of rapamycin could inhibit asecondary immune response in a host that has been pre-immunized with thegene therapy vector or pre-immunized with the protein product encoded bythe trans gene. In preferred embodiments, the present invention relatesto methods and compositions for blocking signal 2, but not signal 1, ofthe interaction between major histocompatibility complex [MHC] onantigen presenting cells [APC] binding to T-cell receptor [TCR], whileat the same time blocking one or more of the co-stimulation pathways:B7-CD28 and CD40-CD40 ligand. Thus, compositions of the presentinvention comprise (1) an agent which blocks signal 2, but not signal I,of the MHCTCR interaction pathway; (2) an agent which blocks aco-stimulation pathway; and (3) a therapeutic agent. The agent whichblocks signal 2, but not signal 1 of the MHC-TCR interaction ispreferably rapamycin, but may also be a rapamycin analog, an antibodywhich binds to the MHC, blocking interaction with TCR, or an antibody toTCR, provided such antibody to TCR is antagonistic, and does notactivate the T cell to which it binds. The agent which blocks aco-stimulation pathway is preferably selected from the group consistingof CTLA4-Ig, antibodies to B7-1, antibodies to B7-2, antibodies to CD28,and antibodies to CD40L. One antibody to CD40L which may be used in thepresent invention as the agent blocking co-stimulation is MRI. Thetherapeutic agent is preferably a gene therapy vector which encodes atherapeutic gene. Suitable gene therapy vectors include viral vectors,such as adenovirus, adeno-associated virus, retrovirus, includinglentivirus vectors. Other gene therapy vectors include cationic oramphiphilic compounds, such as lipids, as well as polymers, liposomesand naked DNA. Useful therapeutic genes include those encoding lysosomalstorage enzymes, such as glucocerebrosidase, alpha-galactosidase A,sphingomyelinase, iduronate sulfatase, alpha-glucosidase,galactosamine-6-sulfatase, beta-galactosidase, galactosamine-4 sulfatase(arylsulfatase B), alpha-glucosidase, and alpha-iduronidase. Otherpreferred therapeutic genes include those useful for the production ofhemophilic proteins, most preferably Factor VIIA, Factor VII and FactorIX. In other embodiments, the therapeutic agent may be a polypeptide, ora combination of polypeptide and gene therapy vectors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the anti-a-gal antibody titers for experiments in which Ad2was co-administered with anti-CD40L and/or Rapamycin.

FIG. 2 shows the anti-α-Ad2 antibody titers for experiments in which Ad2was co-administered with anti-CD40L and/or Rapamycin.

FIG. 3 shows the transgene expression for experiments in which Ad2 wasco administered with anti-CD40L and/or Rapamycin.

FIG. 4 shows the anti-α-gal antibody titers for experiments in which Ad2was co-administered with anti-B7-1 and anti-B7-2 and/or Rapamycin.

FIG. 5 shows the anti-α-Ad2 antibody titers for experiments in which Ad2was co-administered with anti-B7-1 and anti-B7-2 and/or Rapamycin.

DETAILED DESCRIPTION OF THE INVENTION

Rapamycin is a natural product derived from a soil microorganism, whichwas originally described as an antibiotic and subsequently found topossess some immunosuppressive properties. Rapamycin has recently beenapproved for use in patients for kidney transplantation in combinationwith cyclosporine and corticosteroids. Recent reports in the literatureclaim that rapamycin in combination with co-stimulation blockade caninduce tolerance to allografts in mice.

The present invention provides for transient co-administration ofrapamycin or an analog or derivative thereof, together with a genetherapy vector encoding a transgene. The present invention is directedto inhibiting the immune response of a host to the administered genetherapy vector and encoded trans gene product, thus allowing persistenttransgene expression and repeated administration of the gene therapyproduct to the host. The present invention is also of relevance ingenetic disease patients that mount immune responses to proteinreplacement therapies in which case the present invention provides fortransient co-administration of rapamycin together with proteinreplacement therapy. In a further aspect of the invention,co-administration of rapamycin could inhibit a secondary immune responsein a host that has been pre-immunized with the gene therapy vector orpre-immunized with the protein product encoded by the transgene.

Wherever the present application refers to “rapamycin”, in addition tonaturally occurring forms of rapamycin, the present invention includesthe use of rapamycin analogs and derivatives. Many such analogs andderivatives are known in the art, for example, including but not limitedto those described in U.S. Pat. Nos. 6,015,809; 6,004,973; 5,985,890;5,955,457; 5,922,730; 5,912,253; 5,780,462; 5,665,772; 5,637,590;5,567,709; 5,563,145; 5,559,122; 5,559,120; 5,559,119; 5,559,112;5,550,133; 5,541,192; 5,541,191; 5,532,355; 5,530,121; 5,530,007;5,525,610; 5,521,194; 5,519,031; 5,516,780; 5,508,399; 5,508,290;5,508,286; 5,508,285; 5,504,291; 5,504,204; 5,491,231; 5,489,680;5,489,595; 5,488,054; 5,486,524; 5,486,523; 5,486,522; 5,484,791;5,484,790; 5,480,989; 5,480,988; 5,463,048; 5,446,048; 5,434,260;5,411,967; 5,391,730; 5,389,639; 5,385,910; 5,385,909; 5,385,908;5,378,836; 5,378,696; 5,373,014; 5,362,718; 5,358,944; 5,346,893;5,344,833; 5,302,584; 5,262,424; 5,262,423; 5,260,300; 5,260,299;5,233,036; 5,221,740; 5,221,670; 5,202,332; 5,194,447; 5,177,203;5,169,851; 5,164,399; 5,162,333; 5,151,413; 5,138,051; 5,130,307;5,120,842; 5,120,727; 5,120,726; 5,120,725; 5,118,678; 5,118,677;5,100,883; 5,023,264; 5,023,263; 5,023,262; all of which areincorporated herein by reference.

In the sections which follow, detailed therapeutic regimens are providedfor combination therapy of eight specific LSDs (i.e. Gaucher's, Fabry's,Niemann-Pick B, Hunter's, Morquio's, Maroteaux-Lamy, Pompe's, andHurler's-Scheie's in its various clinical manifestations), as well ashemophilic factors Factor VI IA, Factor VIII and Factor IX.

1. Gaucher's

Gaucher's disease is caused by inactivation of glucocerebrosidase andaccumulation of glucocerebroside.

2. Fabry's

Fabry's disease is caused by inactivation of alpha-galactosidase A andaccumulation of GL-3. The enzymatic defect leads to systemic depositionof glycosphingolipids with terminal alpha-galactosyl moieties,predominantly globotriaosylceramide and, to a lesser extent,galabiosylceramide and blood group B substances. In addition to assayfor specific activity of alpha-galactosidase A and accumulation of GL-3,assay for deposition of glycosphingolipid substrates in body fluids andin lysosomes of vascular endothelial, perithelial and smooth musclecells of blood vessels. Other manifestations which can be useful forassay include proteinuria and other signs of renal impairment, such asred cells or lipid globules in the urine, and elevated erythrocytesedimentation rate. Also, anemia, decreased serum iron concentration,high concentration of beta-thromboglobulin, and elevated reticulocytecounts or platelet aggregation. Desnick et al., in Scriver et al.,Metabolic and Molecular Bases of Inherited Disease, (7th ed. 1995) p.2741-2784.

3. Niemann-Pick B

Niemann-Pick B disease is caused by inactivation of sphingomyelinase andaccumulation of sphingomyelin.

4. Hunter's

Hunter's disease (a.k.a. MPSII) is caused by inactivation of iduronatesulfatase and accumulation of dermatan sulfate and heparan sulfate.Hunter's disease presents clinically in severe and mild forms.

5. Morquio's

Morquio's syndrome (a.k.a. MPS IV) results from accumulation of keratansulfate due to inactivation of either of two enzymes. In MPS IVA theinactivated enzyme is galactosamine-'6sulfatase and in MPS IVB theinactivated enzyme is beta-galactosidase.

6. Maroteaux-Lamy

Maroteaux-Lamy syndrome (a.k.a. MPS VI) is caused by inactivation ofgalactosamine4-sulfatase (arylsulfatase B) and accumulation of dermatansulfate.

7. Pompe's

Pompe's disease is caused by inactivation of alpha-glucosidase andaccumulation of glycogen. Hers first proposed the concept of inbornlysosomal disease based on his studies of type II glycogen storagedisease (a.k.a. Pompe's disease, GAA or acid maltase deficiency (AMD);see H. G. Hers, 1965, Gastroenterology 48, 625). An assay foraccumulated intra lysosomal accumulation of glycogen granules,particularly in myocardium, liver and muscle fibers, or serum elevationof CK is described in Hirschorn, in Scriver et al., Metabolic andMolecular Bases of Inherited Disease, (7^(th) ed. 1995) p. 2443-2464.

8. Hurler's-Scheie's

Hurler's-Scheie's disease, also known as MPS1, is caused by inactivationof alpha-iduronidase and accumulation of dermatan sulfate and heparinsulfates, In addition to enzyme assay or by accumulation of the dermatanand heparan sulfates, assay for the disease can be by excessive urinarydermatan and heparan sulfate excretion. Nuefeld and Muenzer, in Scriveret al., Metabolic and Molecular Bases of Inherited Disease, (7th ed.1995) p. 2465-2494.

9. Hemophilic Factors

Other preferred transgenes included genes encoding Factor VIIA, FactorVII or Factor IX. The Factor VIII gene may be full-length (see, e.g.,U.S. Pat. No. 4,965,199; U.S. Pat. No. 5,618,789); B-domain deleted(see, e.g., U.S. Pat. No. 4,868,112 and U.S. Pat. No. 5,661,008) or achimeric hybrid (see, e.g., U.S. Pat. No. 5,563,045; U.S. Pat. No.5,888,974 and U.S. Pat. No. 5,859,204). The Factor IXgene is preferablyof human origin (see, e.g., U.S. Pat. No. 4,994,371 and U.S. Pat. No.5,521,070). The Factor VIIA gene is preferably of human origin (see,e.g., U.S. Pat. No. 4,456,591; U.S. Pat. No. 4,784,950; U.S. Pat. No.5,190,919; U.S. Pat. No. 5,254,672 and U.S. Pat. No. 6,039,944).

Other preferred transgenes include full length cystic fibrosistransmembrane receptor (CFTR), dystrophin, ornithine transcarbamylase(OTC), alpha.1I-antitrypsin (AlAT), Rb, and p53.

Adenoviral vectors are attractive vehicles for gene transfer to a widevariety of dividing and non-dividing cells in vivo, including liver,muscle, lung, brain, heart, etc. However, transgene expression isusually transient in nature due to the generation of cellular andhumoral immune responses to both Ad vector proteins and transgeneproducts. The immune response to adenoviral vector, encoded proteins canbe reduced or circumvented by using deleted partially adenoviral vectorsor pseudoadenoviral vectors (PAV) that are completely deleted ofadenoviral genes (also referred to as fully deleted Ad vectors,mini-adenoviral vectors, helper dependent Ad vectors or gutless Advectors). However, the problem remains of neutralizing antibodies to Adcapsid proteins that prevent re-administration of Ad vector of the sameserotype. Adenoviral vectors, such as pseudoadenoviral vectors,retroviral vectors, adeno associate virus (AAV) vectors or lentiviralvectors do not encode any viral proteins, however, this does not addressthe issue of immunogenicity of the trans gene product, which couldpotentially be a neo-antigen in patients with genetic disease that wewish to treat with these vectors.

Viral Vectors

One of the most frequently used methods of administration of genetherapy, both in vivo and ex vivo, is the use of viral vectors fordelivery of the gene. Many species of virus are known, and many havebeen studied for gene therapy purposes. The most commonly used viralvectors include those derived from adenoviruses, adeno-associatedviruses [AAV) and retroviruses, including lentiviruses, such as humanimmunodeficiency virus [HIV].

Adenovirus

Adenoviral vectors for use to deliver transgenes to cells forapplications such as in vivo gene therapy and in vitro study and/orproduction of the products of trans genes, commonly are derived fromadenoviruses by deletion of the early region 1 (E1) genes (Berkner, K.L., Curro Top. Micro. Immunol. 158:39-66, 1992). Deletion of E1 genesrenders such adenoviral vectors replication defective and significantlyreduces expression of the remaining viral genes present within thevector. However, it is believed that the presence of the remaining viralgenes in adenoviral vectors can be deleterious to the transfected cellfor one or more of the following reasons: (1) stimulation of a cellularimmune response directed against expressed viral proteins, (2)cytotoxicity of expressed viral proteins, and (3) replication of thevector genome leading to cell death.

One solution to this problem has been the creation of adenoviral vectorswith deletions of various adenoviral gene sequences. In particular,partially deleted adenoviral vectors [“DeAd” vectors), andpseudoadenoviral vectors (PAVs), also known as ‘gutless adenovirus’ ormini adenoviral vectors, are adenoviral vectors derived from the genomeof an adenovirus that contain minimal cis-acting nucleotide sequencesrequired for the replication and packaging of the vector genome andwhich can contain one or more transgenes (See, U.S. Pat. No. 5,882,877which covers pseudoadenoviral vectors (PAV) and methods for producing PA V, incorporated herein by reference). Such PAVs or DeAd vectors, whichcan accommodate up to about 36 kb of foreign nucleic acid, areadvantageous because the carrying capacity of the vector is optimized,while the potential for host immune responses to the vector or thegeneration of replication-competent viruses is reduced. P A V and DeAdvectors contain the 5′ inverted terminal repeat (ITR) and the 3′ ITRnucleotide sequences that contain the origin of replication, and thecis-acting nucleotide sequence required for packaging of the adenoviralgenome, and can accommodate one or more trans genes with appropriateregulatory elements, e.g. promoters, enhancers, etc.

Other adenoviral vectors have been created with the deletion of certainspecific genes, which may be some or all of the adenoviral early genes,including E1, E2a, E2b [terminal peptide and DNA polymerase], E3, mostor all of the E4 genes, and may also include some or all of theadenoviral late genes, L1 through L5.

Adenoviral vectors, such as DeAd vectors and PAVs, have been designed totake advantage of the desirable features of adenovirus which render it asuitable vehicle for delivery of nucleic acids to recipient cells.Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about36 kb, which has been well-characterized through studies in classicalgenetics and molecular biology (Hurwitz, M. S., Adenoviruses Virology,3rd edition, Fields et al., eds., Raven Press, New York, 1996; Hitt, M.M. et al., Adenovirus Vectors, The Development of Human Gene Therapy,Friedman, T. ed., Cold Spring Harbor Laboratory Press, New York, 1999).The viral genes are classified into early (designated E1-E4) and late(designated L1-L5) transcriptional units, referring to the generation oftwo temporal classes of viral proteins. The demarcation of these eventsis viral DNA replication. The human adenoviruses are divided intonumerous serotypes (approximately 47, numbered accordingly andclassified into 6 groups: A, B, C, D, E and F), based upon propertiesincluding hemaglutination of red blood cells, oncogenicity, DNA andprotein amino acid compositions and homologies, and antigenicrelationships.

Recombinant adenoviral vectors have several advantages for use as genedelivery vehicles, including tropism for both dividing and non-dividingcells, minimal pathogenic potential, ability to replicate to high titerfor preparation of vector stocks, and the potential to carry largeinserts (Berkner, K. L., Curr. Top. Micro. Immunol. 158:39-66, 1992;Jolly, D., Cancer Gene Therapy 1:51-64, 1994).

PAVs and DeAd vectors have been designed to take advantage of thedesirable features of adenovirus which render it a suitable vehicle forgene delivery. While adenoviral vectors can generally carry inserts ofup to 8 kb in size by the deletion of regions which are dispensable forviral growth, maximal carrying capacity can be achieved with the use ofadenoviral vectors containing deletions of most viral coding sequences,including PAVs. See U.S. Pat. No. 5,882,877 of Gregory et al.; Kochaneket al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Parks et al.,Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996; Lieber et al., J.Virol. 70:89448960, 1996; Fisher et al., Virology 217:11-22, 1996; U.S.Pat. No. 5,670,488; PCT Publication No. WO 96/33280, published Oct. 24,1996; PCT Publication No. WO 96/40955, published Dec. 19, 1996; PCTPublication No. WO 97/25446, published Jul. 19, 1997; PCT PublicationNo. WO 95/29993, published Nov. 9, 1995; PCT Publication No. WO97/00326, published Jan. 3, 1997; Morral et al., Hum. Gene Ther.10:2709-2716, 1998.

Since P A V s and DeAd vectors are deleted for part or most of theadenovirus genome, production of PAVs requires the furnishing ofadenovirus proteins in trans which facilitate the replication andpackaging of a PAV genome into viral vector particles. Most commonly,such proteins are provided by infecting a producer cell with a helperadenbvirus containing the genes encoding such proteins. However, suchhelper viruses are potential sources of contamination of a PAV or DeAdvirus stock during purification and can pose potential problems whenadministering the adenovirus to an individual if the contaminatinghelper adenovirus can replicate and be packaged into viral particles.

It may be advantageous to increase the purity of a PAV or DeAd virusstock by reducing or eliminating any production of helper vectors whichcan contaminate preparation. Several strategies to reduce the productionof helper vectors in the preparation of a DeAd virus or P A V stock aredisclosed in U.S. Pat. No. 5,882,877, issued Mar. 16, 1999; U.S. Pat.No. 5,670,488, issued Sep. 23, 1997 and International Patent ApplicationNo. PCT/US99/03483, incorporated herein by reference. For example, thehelper vector may contain mutations in the packaging sequence of itsgenome to prevent its packaging, an oversized adenoviral genome whichcannot be packaged due to size constraints of the virion, or a packagingsignal region with binding sequences that prevent access by packagingproteins to this signal which thereby prevents production of the helpervirus.

Other strategies include the design of a helper virus with a packagingsignal flanked by the excision target site of a recombinase, such as theCre-Lox system (Parks et al., Proc. Natl. Acad. Sci. USA93:13565-13570,1996; Hardy et al., J. Virol. 71:1842-1849,1997,incorporated herein by reference). Such helper vectors reduce the yieldof wild-type levels.

Another hurdle for DeAd virus or PAV manufacturing, aside from theproblems with obtaining helper vector-free stocks, is that theproduction process is initiated by DNA transfections of the DeAd virusor PAV genome and the helper genome into a suitable cell line, e.g., 293cells. After cytopathic effects are observed in the culture indicating asuccessful infection, which may take up to from 2 to 6 days, the cultureis harvested and is passaged onto a new culture of cells. This processis repeated for several additional passages, up to 7 times more, toobtain a modest cell lysate containing the PAV or DeAd vector and anycontaminating helper vector. See Parks et al., 1996, Proc. Natl. Acad.Sci. USA 93:13565-13570; Kochanek et al., 1996, Proc. Natl. Acad. Sci.USA 93:5731-5736. This lengthy process is not optimal for commercialscale manufacturing. Additionally, this process facilitatesrecombination and rearrangement events resulting in the propagation ofPAV or DeAd viral genomes with unwanted alterations. The use ofadenoviruses for gene therapy is described, for example, in U.S. Pat.No. 5,882,877; U.S. patent, the disclosures of which are incorporatedherein by reference.

Adeno-Associated Virus [AAV]

Adeno-associated virus (AAV) is a single-stranded human DNA parvoviruswhose genome has a size about of 4.6 kb. The AA V genome contains twomajor genes: the rep gene, which codes for the rep proteins (Rep 76, Rep68, Rep 52 and Rep 40) and the cap gene, which codes for AAV structuralproteins (VP-1, VP-2 and VP-3). The rep proteins are involved in AAVreplication, rescue, transcription and integration, while the capproteins form the AAV viral particle. AAV derives its name from itsdependence on an adenovirus or other helper virus (e.g., herpes virus)to supply essential gene products that allow AAV to undergo a productiveinfection, i.e., reproduce itself in the host cell. In the absence ofhelper virus, AAV integrates as a provirus into the host cell'schromosome, until it is rescued by superinfection of the host cell witha helper virus, usually adenovirus (Muzyczka, Curr. Top. Micro. Immunol.158:97-127, 1992).

Interest in MV as a gene transfer vector results from several uniquefeatures of its biology. At both ends of the MV genome is a nucleotidesequence known as an inverted terminal repeat (ITR), which contains thecis-acting nucleotide sequences required for virus replication, rescue,packaging and integration. The integration function of the ITR mediatedby the rep protein in trans permits the MV genome to integrate into acellular chromosome after infection, in the absence of helper virus.This unique property of the virus has relevance to the use of AAV ingene transfer, as it allows for a integration of a recombinant MVcontaining a gene of interest into the cellular genome. Therefore,stable genetic transformation, ideal for many of the goals of genetransfer, may be achieved by use of rAAV vectors. Furthermore, the siteof integration for MV is well-established, and has been localized tochromosome 19 of humans (Kotin et al., Proc. Natl. Acad. Sci.87:2211-2215,1990). This predictability of integration site reduces thedanger of random insertional events into the cellular genome that mayactivate or inactivate host genes or interrupt coding sequences,consequences that can limit the use of vectors whose integration israndom, e.g., retroviruses. However, because the rep protein mediatesthe integration of M V, removal of this gene in the design of rAAVvectors may result in the altered integration patterns that have beenobserved with rMV vectors (Ponnazhagan et al., Hum. Gene Ther.8:275-284,1997).

There are other advantages to the use of MV for gene transfer. The hostrange of AAV is broad. Moreover, unlike retroviruses, MV can infect bothquiescent and dividing cells. In addition, AAV has not been associatedwith human disease, obviating many of the concerns that have been raisedwith retrovirus-derived gene transfer vectors.

Standard approaches to the generation of recombinant rAAV vectors haverequired the coordination of a series of intracellular events:transfection of the host cell with an rAAV vector genome containing atrans gene of interest flanked by the AAV ITR sequences, transfection ofthe host cell by a plasmid encoding the genes for the AA V rep and capproteins which are required in trans, and infection of the transfectedcell with a helper virus to supply the non-AAV helper functions requiredin trans (Muzyczka, N., Curf. Top. Micro. Immunol. 158: 97-129, 1992).The adenoviral (or other helper virus) proteins activate transcriptionof the AAV rep gene, and the rep proteins then activate transcription ofthe AAV cap genes. The cap proteins then utilize the ITR sequences topackage the rAAV genome into an rAAV viral particle. Therefore, theefficiency of packaging is determined, in part, by the availability ofadequate amounts of the structural proteins, as well as by theaccessibility of any cis-acting packaging sequences required in the rAAVvector genome.

One of the potential limitations to high level rAAV production derivesfrom limiting quantities of the AAV helper proteins required in transfor replication and packaging of the rAAV genome. Some approaches toincreasing the levels of these proteins have included placing the AAVrep gene under the control of the HIV LTR promoter to increase repprotein levels (Flotte, F. R. et al., Gene Therapy 2:29-37, 1995); theuse of other heterologous promoters to increase expression of the AAVhelper proteins, specifically the cap proteins (Vincent et al., J.Virol. 71:1897-1905,1997); and the development of cell lines thatspecifically express the rep proteins (Yang, Q. et al., J. Virol.68:4847-4856, 1994).

Other approaches to improving the production of rAAV vectors include theuse of helper virus induction of the AAV helper proteins (Clark et al.,Gene Therapy 3:1124-1132, 1996) and the generation of a cell linecontaining integrated copies of the rMV vector and AAV helper genes sothat infection by the helper virus initiates rAAV production (Clark etal., Human Gene Therapy 6:1329-1341,1995).

rAAV vectors have been produced using replication-defective helperadenoviruses which contain the nucleotide sequences encoding the rAAVvector genome (U.S. Pat. No. 5,856,152 issued Jan. 5, 1999) or helperadenoviruses which contain the nucleotide sequences encoding the AAVhelper proteins (PCT International Publication WO95/06743, publishedMar. 9, 1995). Production strategies which combine high level expressionof the AAV helper genes and the optimal choice of cis-acting nucleotidesequences in the rAAV vector genome have been described (PCTInternational Application No. WO97/09441 published Mar. 13, 1997).

Current approaches to reducing contamination of rAAV vector stocks byhelper viruses, therefore, involve the use of temperature-sensitivehelper viruses (Ensinger et al., J. Virol. 10:328-339, 1972), which areinactivated at the non-permissive temperature. Alternatively, thenon-AAV helper genes can be sub cloned into DNA plasmids which aretransfected into a cell during rAAV vector production (Salvetti et al.,Hum. Gene Ther. 9:695-706,1998; Grimm et al., Hum. Gene Ther.9:2745-2760,1998). The use of AAV for gene therapy is described, forexample, in U.S. Pat. No. 5,753,500; U.S. patent, the disclosures ofwhich are hereby incorporated herein by reference.

Retroviruses

Retrovirus vectors are a common tool for gene delivery (Miller, Nature(1992) 357:455-460). The ability of retrovirus vectors to deliver anunrearranged, single copy gene into a broad range of rodent, primate andhuman somatic cells makes retroviral vectors well suited fortransferring genes to a cell.

Retroviruses are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. This integrated DNAintermediate is referred to as a provirus. Transcription of the provirusand assembly into infectious virus occurs in the presence of anappropriate helper virus or in a cell line containing appropriatesequences enabling encapsidation without coincident production of acontaminating helper virus. A helper virus is not required for theproduction of the recombinant retrovirus if the sequences forencapsidation are provided by co-transfection with appropriate vectors.

Another useful tool for producing recombinant retroviral vectors arepackaging cell lines which supply in trans the proteins necessary forproducing infectious virions, but those cells are incapable of packagingendogenous viral genomic nucleic acids (Watanabe & Temin, Molec. Cell.Biol. (1983) 3(12):2241-2249; Mann et al., Cell (1983) 33:153-159;Embretson & Temin, J. Viroi. (1987) 61 (9):2675-2683). One approach tominimize the likelihood of generating RCR in packaging cells is todivide the packaging functions into two genomes, for example, one whichexpresses the gag and pol gene products and the other which expressesthe env gene product (Bosselman et al., Molec. Cell. Biol. (1987)7(5):1797-1806; Markowitz et al., J. Viroi. (1988) 62(4): 1120-1124;Danos & Mulligan, Proc. Natl. Acad. Sci. (1988) 85:6460-6464). Thatapproach minimizes the ability for co-packaging and subsequent transferof the two-genomes, as Well as significantly decreasing the frequency ofrecombination due to the presence of three retroviral genomes in thepackaging cell to produce RCR.

In the event recombinants arise, mutations (Danos & Mulligan, supra) ordeletions (Boselman et al., supra; Markowitz et al., supra) can beconfigured within the undesired gene products to render any possiblerecombinants non-functional. In addition, deletion of the 3′ LTR on bothpackaging constructs further reduces the ability to form functionalrecombinants.

The retroviral genome and the proviral DNA have three genes: the gag,the pol, and the env, which are flanked by two long terminal repeat(LTR) sequences. The gag gene encodes the internal structural (matrix,capsid, and nucleocapsid) proteins; the pol gene encodes theRNA-directed DNA polymerase (reverse transcriptase) and the env geneencodes viral envelope glycoproteins. The 5′ and 3′ LTRs serve topromote transcription and polyadenylation of the virion RNAs. The LTRcontains all other cis-acting sequences necessary for viral replication.Lentiviruses have additional genes including vit vpr, tat, rev, vpu,nef, and vpx (in HIV-1, HIV-2 and/or SIV). Adjacent to the 5′ LTR aresequences necessary for reverse transcription of the genome (the tRNAprimer binding site) and for efficient encapsidation of viral RNA intoparticles (the Psi site). If the sequences necessary for encapsidation(or packaging of retrovirual RNA into infectious virions) are missingfrom the viral genome, the result is a cis defect which preventsencapsidation of genomic RNA. However, the resulting mutant is stillcapable of directing the synthesis of all virion proteins.

Lentiviruses are complex retroviruses which, in addition to the commonretroviral genes gag, pol and env, contain other genes with regulatoryor structural function. The higher complexity enables the lentivirus tomodulate the life cycle thereof, as in the course of latent infection. Atypical lentivirus is the human immunodeficiency virus (HIV, theetiologic agent of AIDS. In vivo, HIV can infect terminallydifferentiated cells that rarely divide, such as lymphocytes andmacrophages. In vitro, HIV can infect primary cultures ofmonocyte-derived macrophages (MDM) as well as HeLa-Cd4 or T lymphoidcells arrested in the cell cycle by treatment with aphidicolin or gammairradiation. Infection of cells is dependent on the active nuclearimport of HIV preintegration complexes through the nuclear pores of thetarget cells. That occurs by the interaction of multiple, partlyredundant, molecular determinants in the complex with the nuclear importmachinery of the target cell. Identified determinants include afunctional nuclear localization signal (NLS) in the gag matrix (MA)protein, the karyophilic virion-associated protein, vpr, and aC-terminal phosphotyrosine residue in the gag MA protein. The use ofretroviruses for gene therapy is described, for example, in U.S. Pat.No. 6,013,516; and U.S. Pat. No. 5,994,136, the disclosures of which areincorporated herein by reference.

Non-Viral Vectors

Other methods for delivery of DNA to cells do not use viruses fordelivery. These include the use of compounds, such as cationicamphiphilic compounds; as well as DNA in the absence of viral ornon-viral compounds, known as “naked DNA.”

Cationic Amphiphilic Compounds:

Because compounds designed to facilitate intracellular delivery ofbiologically active molecules must interact with both non-polar andpolar environments (in or on, for example, the plasma membrane, tissuefluids, compartments within the cell, and the biologically activemolecule itself), such compounds are designed typically to contain bothpolar and non-polar domains. Compounds having both such domains may betermed amphiphiles, and many lipids and synthetic lipids that have beendisclosed for use in facilitating such intracellular delivery (whetherfor in vitro or in vivo application) meet this definition. Oneparticularly important class of such amphiphiles is the cationicamphiphiles. In general, cationic amphiphiles have polar groups that arecapable of being positively charged at or around physiological pH, andthis property is understood in the art to be important in defining howthe amphiphiles interact with the many types of biologically active(therapeutic) molecules including, for example, negatively chargedpolynucleotides such as DNA.

Examples of cationic amphiphilic compounds that have both polar andnon-polar domains and that are stated to be useful in relation tointracellular delivery of biologically active molecules are found, forexample, in the following references, which contain also usefuldiscussion of (1) the properties of such compounds that are understoodin the art as making them suitable for such applications, and (2) thenature of structures, as understood in the art, that are formed bycomplexing of such amphiphiles with therapeutic molecules intended forintracellular delivery.

(1) Felgner, et al., Proc. Natl. Acad. Sci. USA, 84,7413-7417 (1987)disclose use of positively-charged synthetic cationic lipids includingN->1 (2,3-dioleyloxy) propyl!-N,N,N-trimethylammonium chloride(“DOTMA”), to form lipid/DNA complexes suitable for transfections. Seealso Felgner et al., The Journal of Biological Chemistry, 269(4),2550-2561 (1994).

(2) Behr et al., Proc. Natl. Acad. Sci. USA, 86, 6982-6986 (1989)disclose numerous amphiphiles including dioctadecylamidologlycylspermine(“DOGS”).

(3) U.S. Pat. No. 5,283,185 to Epand et al. describes additional classesand species of amphiphiles including3.beta.>N-(N.sup.1,N.sup.1-dimethylaminoethane) carbamoyl! cholesterol,termed “DC-chol”.

(4) Additional compounds that facilitate transport of biologicallyactive molecules into cells are disclosed in U.S. Pat. No. 5,264,618 toFelgner et al. See also Felgner et al., The Journal Of BiologicalChemistry, 269(4), pp. 2550-2561 (1994) for disclosure therein offurther compounds including “DMRIE” 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide.

(5) Reference to amphiphiles suitable for intracellular delivery ofbiologically active molecules is also found in U.S. Pat. No. 5,334,761to Gebeyehu et al., and in Felgner et al., Methods (Methods inEnzymology), 5, 67-75 (1993).

The use of compositions comprising cationic amphiphilic compounds forgene delivery is described, for example, in U.S. Pat. No. 5,049,386;U.S. Pat. No. 5,279,833; U.S. Pat. No. 5,650,096; U.S. Pat. No.5,747,471; U.S. Pat. No. 5,767,099; U.S. Pat. No. 5,910,487; U.S. Pat.No. 5,719,131; U.S. Pat. No. 5,840,710; U.S. Pat. No. 5,783,565; U.S.Pat. No. 5,925,628; U.S. Pat. No. 5,912,239; U.S. Pat. No. 5,942,634;U.S. Pat. No. 5,948,925; U.S. Pat. No. 6,022,874; U.S. Pat. No.5,994,317; U.S. Pat. No. 5,861,397; U.S. Pat. No. 5,952,916; U.S. Pat.No. 5,948,767; U.S. Pat. No. 5,939,401; and U.S. Pat. No. 5,935,936, thedisclosures of which are hereby incorporated by reference.

“Naked DNA” Transfer

Methods for delivering a non-infectious, non-integrating DNA sequenceencoding a desired polypeptide or peptide operably linked to a promoter,free from association with transfection-facilitating proteins, viralparticles, liposomal formulations, charged lipids and calcium phosphateprecipitating agents is described in U.S. Pat. No. 5,580,859; U.S. Pat.No. 5,963,622; U.S. Pat. No. 5,910,488; the disclosures of which arehereby incorporated by reference.

Combined Viral and Non-Viral Gene Transfer Systems

Gene transfer systems that combine viral and nonviral components havebeen reported. Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:11548; Wu et al. (1994) J. Biol. Chern. 269: 11542; Wagner et al. (1992)Proc. Natl. Acad. Sci. USA 89: 6099; Yoshimura et al., (1993) J. Biol.Chem. 268: 2300; Curiel et al. (1991) Proc. Natl. Acad. Sci USA 88:8850; Kupfer et al. (1994) Hum. Gene Ther. 1437; and Gottschalk et al.(1994) Gene Ther. 1:185. In most cases, adenovirus has been incorporatedinto the gene delivery systems to take advantage of its endosomolyticproperties. The reported combinations of viral and nonviral componentsgenerally involve either covalent attachment of the adenovirus to a genedelivery complex or co-internalization of unbound adenovirus withcationic lipid: DNA complexes.

In a specific example, we have co-administered rapamycin together withAd2/α-gal vector. The rapamycin treatment (70 ug per mouse) was given byI.P. injections daily for 7 days. The Ad2/α-gal vector was deliveredI.V. As seen in the figures, transient treatment with rapamycin was ableto significantly inhibit CTL responses to Ad vector and Ad/α-gal vector.The antibody responses to both Ad vector and a-galactosidase protein wasalso significantly inhibited.

Dosage of Rapamycin

The optimal dosage of rapamycin may be derived by methods known in theclinical arts, including, but not limited to linear equations based onpopulation parameters such as age, weight or sex; non-linear leastsquares modeling methods; Bayesian analysis which employs specific dataabout the medical status of a particular patient; pharmacokineticscompartment modeling; and the trial-and-error method, in which a patientcould be given incremental dosages of rapamycin, and the patient'sreaction thereto could be observed and used to determine the frequencyand quantity of subsequent dosages. Preferred methods for dosing are themethods described in U.S. Pat. No. 5,694,950, the disclosure of which ishereby incorporated herein by reference. In general, a preferred dosageof rapamycin is expected to be from about 0.05 to about 0.15 milligramsof rapamycin per kilogram of the patient's body weight. This dosage maybe increased in order to obtain additional efficacy.

The dosage of gene vector may be determined by one who is expert in thefield. For viral gene therapy, the dosage will generally be in thepreferred ranges from about 10e9 to about 10e13 particles per kg/bodyweight for adenovirus; from about 10e9 to about 10e14 particles perkg/body weight for AAV; and from about 10e6 to about 10e10 transducingunits/kg/body weight for retrovirus or lentivirus.

Rapamycin+Anti-CD40L Experiments

Balb/c mice were injected intravenously With 6e10 particles/mouse ofAd2/CMVα-gal vector on day 0. Mice were given intraperitoneal injectionsof 500 ug/mouse of MR-1, an anti-CD40L antibody, on days −1, +2, +7 and+13. Rapamycin was injected I.P. at 2.5 mg/kg daily from day 0 to +13.One control group of mice received Ad2/CMVα-gal vector alone. Treatmentgroups received (1) Ad vector+Rapamycin; (2) Ad vector+anti-CD40L; or(3) Ad vector+Rapamycin+anti-CD40L. Results are shown in FIG. 1[anti-α-gal antibody titers] and FIG. 2 [anti-Ad2 antibody titers]. Allgroups of mice were bled at the indicated time points to assay forantibodies to Ad2 and α-galactosidase protein expression levels in theserum. As can be seen from the results, anti-a-gal antibody and anti-Ad2antibody titers were significantly lower in the treatment groups than inthe control group. As seen in FIG. 3, transient treatment withanti-CD40L and Rapamycin led to persistent transgene expression forperiods as long as 160 days.

Rapamycin+Anti-B7 Experiments

Balb/c mice were injected intravenously with 7e10 particles/mouse ofAd2/CMVHIα-gal vector on day 0. Mice were given intraperitonealinjections of 100 ug/mouse of anti-B7-1, and 100 ug/mouse of anti-B7-2on days −1, +2 and +7. Rapamycin was injected I.P. at 2.5 mg/kg dailyfrom day 0 to +13. One control group of mice received Ad2/CMVHIα-galvector alone. Treatment groups received (1) Advector+anti-B7-1+anti-B7-2; or (2) Advector+anti-B7-1+anti-B7-2+Rapamycin. Results are shown in FIG. 3[anti-α-gal antibody] and FIG. 4 [anti-Ad2 antibody titers]. All groupsof mice were bled at the indicated time points to assay for antibodiesto Ad2 and α-galactosidase protein expression levels in the serum. Ascan be seen from the results, anti-α-gal antibody and anti-Ad2 antibodytiters were significantly lower in the treatment groups than in thecontrol group.

The above examples are non-limiting, and are included for illustrativepurposes only. The skilled artisan, having read the disclosure containedherein, will readily appreciate that many modifications, additions andimprovements are possible. Such modifications, additions andimprovements are part of the present invention.

The disclosure of each and every publication mentioned in thisspecification is hereby incorporated by reference for the teachingscontained therein.

1. A method for inhibiting the immune response of a host to a proteinreplacement therapy, said method comprising co-administering the proteinreplacement therapy with an effective amount of rapamycin to said host,wherein the humoral immune response to the protein replacement therapyof the rapamycin-treated host is reduced as compared to a host treatedwith the protein replacement therapy alone.
 2. The method of claim 1,wherein the method further comprises administering an effective amountof an agent which blocks a co-stimulation pathway.
 3. The method ofclaim 2, wherein the agent which blocks a co-stimulation pathway isselected from the group consisting of CTLA4-Ig, antibodies to B7-1,antibodies to B7-2, antibodies to CD28, antibodies to CD40L; andcombinations of the above. 4-12. (canceled)
 13. A method for inhibitingthe immune response of a host to a retrovirus gene therapy vectorcomprising a transgene encoding a protein of interest, said methodcomprising co-administering the gene therapy vector with an effectiveamount of rapamycin to said host, wherein the humoral immune response tothe retrovirus gene therapy vector of the rapamycin-treated host isreduced as compared to a host treated with the vector alone.
 14. Themethod of claim 13, wherein the method further comprises administeringan effective amount of an agent which blocks a co-stimulation pathway.15. The method of claim 14, wherein the agent which blocks aco-stimulation pathway is selected from the group consisting ofCTLA4-Ig, antibodies to B7-1, antibodies to B7-2, antibodies to CD28,antibodies to CD40L; and combinations of the above.
 16. The method ofclaim 13, wherein the retrovirus vector is lentivirus vector.
 17. Themethod of claim 14, wherein the transgene encodes a protein selectedfrom the group consisting of glucocerebrosidase, alpha-galactosidase A,beta-galactosidase, sphingomyelinase, iduronate sulfatase,alpha-glucosidase and alpha-iduronidase.
 18. The method of claim 14,wherein the transgene encodes a protein selected from the groupconsisting of Factor VIIA, Factor VII or Factor IX.
 19. A method forinhibiting the immune response of a host to a non-viral gene therapyvector comprising a transgene encoding a protein of interest, saidmethod comprising co-administering the gene therapy vector with aneffective amount of rapamycin to said host, wherein the humoral immuneresponse to the retrovirus gene therapy vector of the rapamycin-treatedhost is reduced as compared to a host treated with the vector alone. 20.The method of claim 18, wherein the non-viral gene therapy vectorcomprises DNA.
 21. The method of claim 19, wherein the non-viral genetherapy vector further comprises a cationic amphiphilic compound. 22.The method of claim 1, wherein the protein replacement therapy comprisesglucocerebrosidase.
 23. The method of claim 1, wherein the proteinreplacement therapy comprises alpha-galactosidase A.
 24. The method ofclaim 1, wherein the protein replacement therapy comprisessphingomyelinase.
 25. The method of claim 1, wherein the proteinreplacement therapy comprises iduronate sulfatase.
 26. The method ofclaim 1, wherein the protein replacement therapy comprisesgalactosamine-6-sulfatase.
 27. The method of claim 1, wherein theprotein replacement therapy comprises beta-galactosidase.
 28. The methodof claim 1, wherein the protein replacement therapy comprisesgalactosamine-4-sulfatase.
 29. The method of claim 1, wherein theprotein replacement therapy comprises alpha-glucosidase.
 30. The methodof claim 1, wherein the protein replacement therapy comprisesalpha-iduronidase.